RESEARCH ARTICLE

Development of an Amperometric-Based Glucose Biosensor to Measure the Glucose Content of Fruit Lee Fung Ang1, Lip Yee Por2, Mun Fei Yam1* 1 School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800, Penang, Malaysia, 2 Faculty of Computer Science and Information Technology, University of Malaya, 50603, Kuala Lumpur, Malaysia * [email protected]

Abstract

OPEN ACCESS Citation: Ang LF, Por LY, Yam MF (2015) Development of an Amperometric-Based Glucose Biosensor to Measure the Glucose Content of Fruit. PLoS ONE 10(3): e0111859. doi:10.1371/journal. pone.0111859 Academic Editor: Frederik Börnke, Leibniz-Institute for Vegetable and Ornamental Crops, GERMANY Received: May 1, 2014 Accepted: October 2, 2014 Published: March 19, 2015 Copyright: © 2015 Ang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

An amperometric enzyme-electrode was introduced where glucose oxidase (GOD) was immobilized on chitosan membrane via crosslinking, and then fastened on a platinum working electrode. The immobilized enzyme showed relatively high retention activity. The activity of the immobilized enzyme was influenced by its loading, being suppressed when more than 0.6 mg enzyme was used in the immobilization. The biosensor showing the highest response to glucose utilized 0.21 ml/cm2 thick chitosan membrane. The optimum experimental conditions for the biosensors in analysing glucose dissolved in 0.1 M phosphate buffer (pH 6.0) were found to be 35°C and 0.6 V applied potential. The introduced biosensor reached a steady-state current at 60 s. The apparent Michaelis-Menten constant (KMapp ) of the biosensor was 14.2350 mM, and its detection limit was 0.05 mM at s/n > 3, determined experimentally. The RSD of repeatability and reproducibility of the biosensor were 2.30% and 3.70%, respectively. The biosensor was showed good stability; it retained ~36% of initial activity after two months of investigation. The performance of the biosensors was evaluated by determining the glucose content in fruit homogenates. Their accuracy was compared to that of a commercial glucose assay kit. There was no significance different between two methods, indicating the introduced biosensor is reliable.

Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This project was supported by research grant from the Malaysia Toray Science Foundation (MTSF) (Grant No. 304, PFARMASI 650295, T102) and High Impact Research Grant (UM.C/625/1/HIR/ MOHE/FCSIT/15) from the University of Malaya. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.

Introduction Biosensors have wide applications ranging from the food industry to environmental monitoring and clinical analysis. The concept of a biosensor was first introduced by Clark and Lyons [1] in the form of an oxygen electrode for monitoring glucose. An electrochemical biosensor has been defined as “a self-contained integrated device, which is capable of providing specific quantitative and semi-quantitative analytical information using a biological recognition element (biochemical receptor) which is retained in direct spatial contact with an electrochemical transduction element” [2]. It should respond to analytes selectively, continuously, rapidly, specifically and ideally without any added reagent. Enzymes, antibodies, nucleic acids and

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receptors are the four main groups of biological elements encountered in biosensors, with enzymes being the most regularly employed. About half of the published papers on biosensors are related to glucose monitoring, primarily due to its metabolic and medical importance [3–7]. It also serves as a good analyte for the development of new biosensors. In the Clark oxygen electrode, glucose oxidase (GOD) was retained by a perm-selective membrane adjacent to an amperometric detector as the sensing element. GOD, a highly specific enzyme, is the most widely studied of all amperometric-based enzymes for biosensors. This enzyme catalyses the oxidation of glucose to gluconolactone according to the following reaction: bD  glucose þ GODðFADÞ Ð GODðFADH2 Þþ D  gluconodlactone

ðreaction 1Þ

GOD catalyzes the oxidation of β-D-glucose to D-glucono-δ-lactone and hydrogen peroxide (H2O2) using molecular oxygen (O2) as the electron acceptor. The two-stage enzyme process, typical for the class of oxidases, consists of enzymatic oxidation of glucose by its cofactor FAD (flavin adenine dinucleotide) (redox centre) which is then reduced to FADH2 (reaction 1). This is followed by its reoxidation or regeneration of the biocatalyst by O2 with formation of H2O2 (reaction 2): GODðFADH2 Þþ O2 ! GODðFADÞþ H2 O2

ðreaction 2Þ

The D-glucono-δ-lactone produced in the reaction (1.1) is a weak competitive inhibitor of glucose, which hydrolyses spontaneously to gluconic acid (reaction 3). D  gluconodlactone þ H2 O ! gluconic acid

ðreaction 3Þ

The overall reaction is expressed as: GOD

bD  glucose þ O2 þH2 O! gluconicacidþ H2 O2

ðreaction 4Þ

Although specific for β-D-glucose, GOD can be used to measure total glucose because α-glucose is converted to the β-form by mutarotation at equilibrium. Thus, GOD is widely used for the determination of free glucose in body fluids. In amperometry, the detection of glucose is usually based on measuring the increase in the anodic current (H2O2 oxidation) or the decrease in the cathodic current (O2 reduction) at the electrochemical cell at a fixed potential. Oxygen electrode-based glucose biosensors and hydrogen peroxide electrode-based glucose biosensors are two commonly studied amperometric glucose biosensors. The reactions on a cathodically polarized platinum electrode are shown below: Pt

O2 þ 2Hþ þ 2e!H2 O2 Pt

H2 O2 þ 2Hþ þ 2e!2H2 O

ðreaction 5Þ ðreaction 6Þ

The basic principle of the first-generation of glucose biosensors is the quantification of glucose through electrochemical detection of the enzymatically liberated H2O2, where the current produced is proportional to its concentration. In an amperometric glucose biosensor, the working potential over which H2O2 is detected is typically between 500–750 mV vs. Ag/AgCl. There are different procedures for immobilizing the biological component in a thin layer at the transduction surface of electrochemical biosensors. For instance, Low et al. [8] demonstrated the preparation of ferrocene-containing photopolymeric films based on hydrophilic methacrylate polymer, which can prevent leaching of both ferrocene and enzyme. A new glucose

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biosensor based on covalent immobilization of GOD on carbon nanotube film functionalized with carboxylic acid groups was described by Xue et al. [9]. Subsequently, Lim et al. [10] reported a glucose biosensor based on electrochemical co-deposition of palladium nanoparticles and GOD onto a carbon nanotube film. A novel glucose biosensor utilizing nanoporous ZrO2/ chitosan composite film as an immobilization matrix for GOD was developed by Yang et al. [11]. Another technique involving Langmuir-Blodgett film deposition of a conducting organic polymer poly (3-dodecyl thiophene) to immobilize GOD for glucose biosensing was reported by Singhal et al. [12]. The properties of immobilized enzymes are governed by the properties of both the enzyme and the support material. Generally, the support material should possess some of the desirable characteristics such as high affinity to proteins, ease of chemical modifications, hydrophilicity, mechanical stability and rigidity, possibility of regeneration so as to provide the system with a permeable surface suitable for a chosen biotransformation [13]. In recent years, chitosan has been widely used as a support for enzyme immobilization in the construction of biosensors [14–20]. Chitosan is poly[-(1–4)-2-amino-2-deoxy-ß-D-glucopyranose], a cationic polysaccharide abundant in the shells of crustaceans. It is derived by partial deacetylation of chitin [21]. The presence of amino groups gives it a basic character. It is an ideal immobilization matrix for the fabrication and construction of biosensors, with properties including excellent membraneforming ability, high water permeability, good adhesion, biocompatibility, biodegradability, antibacterial properties, lack of toxicity, heavy metal ion chelation, hydrophilicity and a remarkable affinity for proteins due to the presence of reactive amino and hydroxyl functional groups [13,20,22]. Its ability to absorb metal ions and various organic halogen substances can also prevent the immobilized enzyme used in biosensors from damage [23]. In addition, chitosan can form a thermally and chemically inert film that is insoluble in water [23]. The sugar (fructose, sucrose and glucose) content in ripened fruits is correlated with their glycaemic index. Diabetic patients often question and worry whether it is safe for them to eat fruit, which can contain large quantities of sugar. Glucose is a major monosaccharide found in almost all fruits and is easily absorbed through gastrointestinal tract to increase blood glucose levels. Thus, the glucose content attracts great attention as an indicator of the glycaemic index for fruits. Therefore, the development of simple, reliable and economical glucose biosensors is desirable for measuring the glucose content of fruits. Application of such biosensor for quality control in the fruit industry can have an economic impact as well as health benefits to end users who are diabetic patients. In this paper, we report the fabrication and characterization of an amperometric-based glucose biosensor for measuring glucose content in fruit homogenates.

Materials and Methods 2.1 Materials Glucose oxidase from Aspergillus niger (EC 1.1.3.4, type VII, 185,000 units/g solid), glutaraldehyde (grade II, 25% aqueous solution) and glycerol were purchased from Sigma (St. Louis, USA). Chitosan (Code #22742) was procured from Fluka (Switzerland). D(+)glucose monohydrate was supplied by System (Malaysia). Glacial acetic acid, citric acid monohydrate and potassium phosphate (KH2PO4) were obtained from R&M Marketing (Essex, U.K) while disodium hydrogen phosphate dehydrate (Na2HPO42H2O) and sodium chloride were bought from Hamburg Chemical (Germany). Tri-sodium citrate was purchased from Grauwmeer (Leuven, Belgium). Hydrogen peroxide (>30% w/v) was bought from Fisher Scientific (Loughborough, UK), while premounted dialysis membrane (Ezee-Mount, type “C”) was bought from Fisher Scientific (Pittsburgh, Pennsylvania, USA). Aluminium oxide (highly pure for polishing)

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was purchased from BDH Laboratory Supplies (England). A PK-4 Polishing kit (MF-2060), platinum working electrode (MF-2013), and silver/silver chloride (Ag/AgCl) reference electrode (MF-2079) were purchased from Bioanalytical Systems Inc. (West Lafayette, Indiana, USA). All chemicals were of analytical grade and were used without further purification. The molecular weight of chitosan was determined by dilute solution viscosity using an Ubbelohde viscometer U-tube (size C, VS-220, Technico, England) and the degree of deacetylation was determined using the first derivative UV-spectrophotometric method. The chitosan had viscosity-average molecular weight of 981.80 kDa and was 82.44% deacetylated.

2.2 Methods 2.2.1 Electrochemical measurement. Amperometric detection of glucose was performed using a potentiostat (CV-1B cyclic voltammograph, Bioanalytical Systems Inc. (BAS), West Lafayette, Indiana, USA) poised at +0.6 V connected to an integrator-plotter (D-2500 ChromatoIntegrator, Hitachi, Tokyo, Japan) and a digital multimeter (8022A, Fluke, USA). The conventional three electrodes consisted of a silver/silver chloride (Ag/AgCl) reference electrode (MF2079, BAS, West Lafayette, Indiana, USA), a platinum wire (0.25 mm diameter, 99.99%, Aldrich, Milwaukee, Wisconsin, USA) as counter electrode and a platinum working electrode (MF-2013, BAS, West Lafayette, Indiana, USA) with the enzyme-chitosan layer and a protective dialysis membrane. Unless stated otherwise, all experiments were carried out in 10 ml of phosphate buffer (0.1 M, pH 7.0) maintained at 25 ± 0.1°C using a digital temperature controller (Model 9001, Poly Science, USA) with stirring to provide convective transport. Glucose stock solution (prepared in phosphate buffer) was allowed to mutarotate at 4°C for at least 24 h prior to use, since only β-D-glucose is a substrate for the enzymatic reaction. At stable background current, aliquots of the β-D-glucose stock solution were introduced into the stirred phosphate buffer and the steady anodic current produced by the enzymatically generated H2O2 was recorded. 2.2.2 Preparation of chitosan membrane and characterization. One gram of chitosan was dissolved in 100 ml of 0.8% (w/v) acetic acid and stirred overnight to ensure complete dissolution. Varying volumes of chitosan solution were then pipetted into petri dishes at a premeasured volume per surface area (ml/cm2) and then allowed to dry overnight in an oven at 60°C. The thickness of the membranes was measured using a micrometer (digimatic micrometer, Mitutoyo, Tokyo, Japan) at five locations (the centre and four corners), and the mean thickness calculated. Mechanical properties such as tensile strength and elongation at break were measured with a texture analyser (TA.XT2, Stable Micro System, Haslemere, Surrey, UK) equipped with a 5 kg load cell. The other prepared membranes were neutralized with 1% w/v sodium hydroxide (NaOH) for 30 minutes followed by rinsing with distilled water to remove excess NaOH. The neutralized membranes were cut into squares (1.5 x 1.5 cm2) for the diffusion study and for enzyme immobilization. 2.3.3 Study on the diffusion of hydrogen peroxide through chitosan membranes. The diffusion properties of chitosan membranes cast in different thicknesses were determined by measuring the electrode response to H2O2 using amperometric detection. The anodic current generated by H2O2 which had diffused through the membrane was sensed by (i) a bare platinum electrode (bare PT), (ii) an electrode covered with dialysis membrane (Dialysis PT) and (iii) an electrode covered with blank chitosan membrane in addition to a dialysis membrane (CHIT/PT). A comparison of the effect of the chitosan membranes on the electrode response to H2O2 was then made. 2.2.4 Enzyme immobilization. The method of Magalhães et al. [24] was modified to immobilize the GOD onto the chitosan membrane. One side of the square of membrane (with a

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thickness of 0.35 ml/cm2) was coated with 20 μl of 1% (v/v) glutaraldehyde and allowed to dry at room temperature. Subsequently, 20 μl of 10 mg/ml (0.2 mg) GOD in phosphate buffer (0.1 M, pH 7.0) containing 5% (v/v) glycerol was spread evenly onto the same surface of the membrane with the aid of an L-shaped rod. The immobilized membrane was then left to dry at room temperature. The small amount of glycerol in the enzyme solution acts as an emollient to facilitate even spreading of GOD on the membrane surface. The dried membrane was washed with distilled water and kept in phosphate buffer (0.1 M, pH 7.0) at 4°C until further use. 2.2.5 Construction of the glucose biosensor. The platinum electrode was first polished with 0.05 μm alumina on a polishing pad, washed with distilled water and finally sonicated for 2 minutes to remove the alumina particles. Then the GOD-chitosan membrane and a moist dialysis membrane as a lamination layer were fastened onto the surface of the platinum electrode with an O-ring. 2.2.6 Optimization of experimental variables for the analysis of glucose using biosensor. The factors influencing enzymatic activity and ultimately biosensor performance were investigated. These included applied potential, membrane thickness, glutaraldehyde concentration, enzyme concentration, temperature, pH and buffer concentration. The effect of applied potential on the steady-state current response of the enzyme-electrode in the potential range from 0.30–0.80 V in 0.05 V increments was studied. The potential was set at the lowest voltage of 0.30 V and the background current allowed to decay to a steadystate value before increasing the applied potential stepwise to 0.80 V. A comparison of the response current at different applied potentials generated by the bare platinum electrode in phosphate buffer and in 0.05 mM H2O2 in phosphate buffer as well as by the biosensor (GODCHIT/PT) in 2 mM glucose in phosphate buffer was made. The fabricated chitosan membranes were cast in thicknesses ranging from 0.21 to 0.42 ml/ cm2. Glutaraldehyde concentration was investigated from 0.1% v/v to 1.0% v/v. Various amounts of GOD (0.05–0.8 mg) in phosphate buffer (0.1 M, pH 7.0) were immobilised on the chitosan membrane. The effect of the temperature of analysis (from 15–50°C) on biosensor performance was studied by measuring the anodic current generated. The optimal pH for enzymatic activity was investigated by varying the pH value from 4.0–8.0. Buffers at different pH values were prepared with 0.1 M citrate buffer to obtain pH values from 4.0–5.5 and with 0.1 M phosphate buffer to attain pH values in the 6.0–8.0 range. The concentration of the working buffer at optimum pH was investigated from 0.01–0.20 M. The optimal value obtained for each parameter was used in subsequent experiments. 2.2.7 Calibration of the glucose biosensor. Six GOD-CHIT/PT electrodes were prepared according to the optimal conditions: 0.6 mg GOD was immobilized onto 0.21 ml/cm2 chitosan membrane and crosslinked with 0.2% v/v glutaraldehyde. The biosensors performed their electrochemical measurements at 35°C, in a supporting electrolyte of 0.1 M phosphate buffer, pH 6.0. Aliquots of β-D-glucose stock solution were successively added into 10 ml of stirred phosphate buffer in an electrochemical cell. Three different glucose stock solutions with concentrations of 0.01 M, 0.1 M and 1.0 M were prepared to obtain the hydrodynamic response for glucose from 0.01 to 130 mM. The mean value of the anodic current (μA) was plotted against the glucose concentration (in mM). The linear range of the biosensor was then determined from the saturation curve. The detection limit at which the signal to noise (s/n) > 3 was determined experimentally. The apparent Michaelis-Menten constant, KMapp and Imax were determined by from a Eadie-Hofstee plot using the equation shown below: I ¼ Imax  KMapp

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 I c

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where I is the steady state current after the addition of substrate, c is the bulk concentration of the substrate (glucose) and Imax is the maximum current measured under a saturated substrate condition. The KMapp and Imax of the biosensor were then determined from the slope and intercept on the y-axis of the plot. 2.2.8 Repeatability and reproducibility. The repeatability generated by the glucose biosensor was studied by measuring the anodic current generated by 3.98 mM glucose in 10 ml phosphate buffer (pH 6.0) a total of 20 times in a single day. On the other hand, the reproducibility of the biosensors was studied by measuring the current generated by 3.98 mM glucose in 10 ml phosphate buffer (pH 6.0) by using six different glucose biosensors. Each biosensor was tested by replicate (n = 3) analysis. The total mean value was calculated and the relative standard deviation (RSD) provided the analytical precision. The RSD was calculated using the following equation: RSDð%Þ ¼

Standard deviation  100% Average

2.2.9 Storage stability study. Three good GOD-CHIT/PT biosensors were prepared. On the other hand, three free enzyme-electrodes (GOD/PT) were prepared with the same amount of GOD, where the enzyme was coated directly on the platinum electrode surface without immobilization, only protected with a layer of dialysis membrane and fastened with an O-ring. The storage stability of the two types of enzyme-electrodes was explored under optimal experimental conditions. The responses of the three GOD-CHIT/PTs and three GOD/PTs to 3.98 mM glucose were measured daily during the first two weeks. After 2 weeks, the biosensors were tested every 3–5 days over a period of 80 days. The mean of the relative current to the initial current sensed by these biosensors was plotted as a function of time. All enzyme electrodes were kept in 0.1 M phosphate buffer (pH 7.0) and stored at 4°C when not in use. 2.2.10 Glucose determination in fruit. Fresh fruits (banana, watermelon, orange, mango, apple and pear) were obtained from the local market and homogenized with distilled water (10 or 1 g in 1 L). Further dilution using distilled water was made if necessary. The homogenate was centrifuged at 3000 rpm and 4°C and the supernatant was filtered through a nylon syringe filter (0.2 μm) prior to determining the glucose content using the glucose biosensor and a commercial glucose assay kit (Sigma-Aldrich, USA) that relies on spectrophotometric detection. The statistical analysis method (T-test) was used to compare the results obtained from the glucose biosensor and the commercial glucose assay kit at a level of significance P

Development of an amperometric-based glucose biosensor to measure the glucose content of fruit.

An amperometric enzyme-electrode was introduced where glucose oxidase (GOD) was immobilized on chitosan membrane via crosslinking, and then fastened o...
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